WO1999058727A1

WO1999058727A1 - Viral characterization by direct detection of capsid proteins

Info

Publication number: WO1999058727A1
Application number: PCT/US1999/010658
Authority: WO
Inventors: John J. Thomas; Catherine Fenselau
Original assignee: University Of Maryland At Baltimore County
Priority date: 1998-05-14
Filing date: 1999-05-14
Publication date: 1999-11-18
Also published as: AU3990799A; US6037118A

Abstract

The present invention provides a method of direct detecting of capsid proteins from intact viral particles using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry, which enables viral proteins to be characterized at the femtomolar level in complex biological milieu with minimal or no cleanup.

Description

VIRAL CHARACTERIZATION BY DIRECT DETECTION

OF CAPSID PROTEINS

BACKGROUND OF THE INVENTION

Cross Reference to Related Applications

This application claims benefit of non-provisional

application US Serial number 09/120,861 , filed July 22, 1998, which

claims benefit of provisional application US Serial number

60/085,389, filed May 14, 1998, now abandoned.

Field of the Invention

The present invention relates generally to the fields of

analytical chemistry and biochemistry. More specifically, the present

invention relates to direct viral characterization by mas s

spectrometric detection of capsid proteins. Description of the Related Art

Medical, agricultural, and military personnel who need to

monitor and identify microbial agents often utilize enzyme-linked

immunosorbent assays, serological, morphological, and other

microbiological methods (1). Certain viruses represent a biological

threat; therefore, a need exists for rapid detection of characteristic

viral biomarkers. Field responses to biological agents require

completion of the entire analysis in less than 10 minutes, thereby

leaving only minutes for chemical manipulation of the sample.

Since the inception of matrix-assisted laser desorption

ionization (MALDI) mass spectrometry (2), the marriage of this

desorption/ionization technique with time-of-flight mass

spectrometers has enabled scientists to detect large biopolymers with

high sensitivity (3). Semi-purified mixtures of proteins have bee n

analyzed with matrix-assisted laser desorption ionization mas s

spectrometry in the past, thereby providing a more accurate

alternative to the commonly used SDS/PAGE analysis of protein

mixtures (4). The energy imparted from matrix-assisted laser

desorption ionization onto proteins and the conformationally

indiscriminate time-of-flight removes some effects of complex protein

solution behavior, which can alter the gel migration of different proteins. Mass spectrometry also provides mass measurements in a

shorter amount of time than electrophoresis.

Viruses and bacteriophages contain large quantities of

proteins, which perform various functions for each virion. One type

of protein commonly found in viruses, which is known as a

nucleocapsid or coat protein, encapsulates nucleic acid molecules a t

the core of the organism. In certain more complex virions, this

protein capsid layer is surrounded by a lipoglycoprotein layer. The

viruses in this study consist of a defined number of homogeneous

capsid protein molecules in close association with the viral nucleic

acids. Figure 1 displays the structures of the MS2, tobacco mosaic

(TMV) and Venezuelan equine encephalitis (VEE) viruses.

TMV and the MS2 bacteriophage are representative of a n

RNA virus and bacteriophage respectively. Despite different protein

conformations and resulting structural organization, these proteins

perform similar functions of acting as translational repressors of th e

phage-encoded replicase gene, protecting the viral RNA, and serving

as a nucleation site for viral assembly. The icosadeltahedron shaped

MS2 bacteriophage consist of 180 copies of a coat protein with a

molecular weight of 13,730 Da surrounding a single stranded RNA,

and one copy of an "A-protein" (MW -44 kDa). The TMV virus is a spherical virus consisting solely of coat proteins, which has species

dependent molecular weights of 17,100-17,600 Da.

Venezuelan equine encephalitis was used as an example of

membrane-containing viruses, which are assembled by interaction of

viral proteins with the host cell membrane. Venezuelan equine

encephalitis contains a single stranded RNA genome encapsulated b y

240 copies of a nucleocapsid protein (M.W. 30,940 Da) surrounded b y

a lipid bilayer containing two glycoproteins, El and E2 that have

molecular masses of -50,000 and -56,000 Da, respectively (5 -7).

While the relative abundance of these unique biomarkers presents a n

opportunity for identification of the virus, scientists have only

recently attempted to rapidly identify these biomarkers. Previously ,

mass spectrometric detection of viral coat proteins required extensive

off-line or elaborate on-line cleanup processes. Despeyroux et al.

utilized HPLC on-line with electrospray mass spectrometry to

determine molecular weights of specific capsid proteins (8).

The prior art is deficient in the lack of effective means of

identifying viruses from completely crude biological media with

minimal or no cleanup. Further, the prior art is deficient in the lack

of effective means for rapid detection of as little as femtomoles of coat protein within a complex biological medium. The present

invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

Matrix-assisted laser desorption ionization (MALDI) mas s

spectrometry has enabled detection of viral proteins with minimal

separation from crude biological media. This analysis has been

achieved with a combination of organic acids and high energy

desorption with a nitrogen laser. The molecular weights from these

proteins are sufficiently unique to differentiate among species of

viruses in a variety of biological conditions. This analysis has

provided the means for rapid detection of as little as femtomoles of

coat protein within a complex biological medium.

In one embodiment of the present invention, there is

provided a method of detecting viral structural proteins rapidly an d

directly from a complex environment by utilizing mass spectrometry

with the addition of specific organic acids.

In a preferred embodiment, the organic acid is acetic acid

or citric acid, and premixed with mass spectrometry matrix. A representative viral protein is a capsid protein. Preferably, the semi-

purified or non-purified sample is prepared from the complex

environment.

Representative examples of mass spectrometry are fast

atom bombardment, mass spectrometry, plasma desorption mas s

spectrometry, laser desorption mass spectrometry, matrix- assisted

laser desorption ionization time-of-flight mass spectrometry a n d

electrospray mass spectrometry, wherein the laser is of any kind an d

has wavelength of any range. Preferably, the method detects

femtomolar concentration of the viral protein in less than 3 minutes .

The method also detects bacteriophage proteins.

Other and further aspects, features, and advantages of th e

present invention will be apparent from the following description of

the presently preferred embodiments of the invention given for the

purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features ,

advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more

particular descriptions of the invention briefly summarized above

may be had by reference to certain embodiments thereof which are

illustrated in the appended drawings. These drawings form a part of

the specification. It is to be noted, however, that the appended

drawings illustrate preferred embodiments of the invention an d

therefore are not to be considered limiting in their scope.

Figure 1 shows the structure of bacteriophage and virus .

Figure 1 A shows MS2 bacteriophage, Figure IB shows tobacco

mosaic virus, and Figure IC shows Venezuelan equine encephalitis

virus.

Figure 2 shows the matrix-assisted laser desorption

ionization mass spectra of semi-purified MS2 bacteriophage with

acetic acid (Figure 2A), and citric acid (Figure 2B ) with α-cyano-4-

hydroxycinnamic acid matrix .

Figure 3 shows the matrix-assisted laser desorption

ionization mass spectrum of MS2 bacteriophage from crude culture

broth with acetic acid and α-cyano-4-hydroxycinnamic acid matrix.

Figure 4 shows the matrix-assisted laser desorption

ionization mass spectrum of tobacco mosaic virus directly from a piece of an infected leaf. 2M citric acid and sinapinic acid matrix

were applied to the infected leaf piece prior to desorption.

Figure 5 shows MALDI mass spectrum of sucrose

gradient purified Venezuelan equine encephalitis with acetic acid an d

sinapinic acid matrix.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, a method of rapid detecting of

viral proteins from a complex medium is disclosed, comprising th e

steps of preparing a crude sample of the medium; mixing the sample

with an organic acid; and detecting viral characteristic structural

proteins by utilizing mass spectrometry. As used herein, "crude

sample" refers to the unadulterated growth medium which ma y

contain salts, buffers, peptides, lipids, oligonucleotides, sugars an d

other chemicals. Certain organic acids will be useful in this technique

as will be readily recognized by a person having ordinary skill in this

art based on the disclosure herein. Preferably, the organic acid is

acetic acid or citric acid, and premixed with mass spectrometry

matrix. The crude sample is either semi-purified or non-purified preparation and may contain only femtomolar levels of viral proteins .

In a preferred embodiment, the viral protein is a capsid protein.

Representative examples of mass spectrometry are fast atom

bombardment mass spectrometry, plasma desorption mas s

spectrometry, laser desorption mass spectrometry, matrix-assisted

laser desorption ionization time-of-flight mass spectrometry an d

electrospray mass spectrometry, wherein the laser is of any kind an d

has wavelength of any range. In a preferred embodiment, th e

method detects femtomolar concentration of the viral protein in less

than 3 minutes. The method may also detect bacteriophage proteins.

The following examples are given for the purpose of

illustrating various embodiments of the invention and are not me ant

to limit the present invention in any fashion.

EXAMPLE 1

Equipment and Chemicals

MALDI spectra were taken on a Kratos/Shimadzu Kompact

MALDI III ™ (Manchester, U. K.) in linear mode. Matrix-assisted laser

desorption ionization was attained with a nitrogen laser (λ = 337 nm)

and an acceleration voltage of 20 kV. Samples were desorbed from stainless steel plates with preconstructed sample wells. All protein

standards and matrices were purchased from Sigma Chemical Co. (St.

Louis, Mo) and used as received. HPLC-grade solvents were

purchased from Fisher Scientific, Inc. (Pittsburgh, PA).

EXAMPLE 2

Biological Growth Conditions

A host culture of an Hfr strain (i.e. male specific) of E. Coli

(ATCC 15669; Rockville, MD) was grown for 16 hours at 37°C in MS2

broth, and was then used to inoculate a larger culture (9).

Bacteriophage stock suspension was added to the bacterial culture

when the culture reached 10⁸ cfu/ml as determined with a Gilford

RESPONSE™ dual channel UV-spectrophotometer (Gilford Instruments ;

Oberlin, OH). The phage culture was incubated at 37°C for 2-3 hours.

Homogenates of ground tobacco leaf samples (ATCC PV-

635) in 50 mM phosphate buffer (pH 7.5) were used to propagate

tobacco mild green mosaic virus (U2 strain) onto healthy young

tobacco plants. The plants were inoculated by causing microscopic

abrasions with a throat-powder spray of carborundum before applying the sap homogenate by hand (10). The infected plants were

sustained under incandescent/UV light for 2 weeks before harvest.

All materials in contact with TMV were sterilized after use.

Venezuelan equine encephalitis Trinidad donkey virus

(VEE-TRD) 3000 strain was expressed from cDNA, and propagated in

baby hamster kidney cells. The growth media was composed of

EMEM supplemented with 5% fetal bovine serum. The collected virus

was precipitated with polyethylene glycol and salt. The precipitate

was resuspended in phosphate buffer saline and layered onto a

sucrose gradient. The semi-purified virus was irradiated with Co⁶⁰ for

safe handling.

EXAMPLE 3

Analytical Methods

Crude MS2, as well as VEE preparations with 50% ( v/v )

organic acid were applied directly to the stainless steel MALDI slide

with a small volume metal tip syringe, and sandwiched by the matrix

(11). Crude TMV samples were analyzed by cleanly cutting strips of

infected leaves, and strategically placing them on stainless steel slides with one-sided tape. The leaf slices were treated with 0.3 μl of

organic acid 15-30 seconds before adding 2 x 0.3 μl of matrix. Semi-

purified preparations of MS2/E. Coli and TMV were processed b y

pelleting bacteria and plant cell debris with high speed centrifugation ,

and the supernatant containing the virus was partially purified b y

centrifugation-filtration (Ultrafree filters, Millipore Corp., Bedford,

MA).

External calibrations were executed with chicken

lysozyme (M.W. 14,307), bovine trypsinogen (M.W. 23,957), a n d

bovine serum albumin (M.W. 66,431) for MS2, TMV, and VEE-TRD

analysis, respectively. In order to avoid spatial source effects,

calibration for crude TMV analysis was performed by applying th e

calibrant and matrix to a separate piece of tobacco leaf.

EXAMPLE 4

Results

Detection, mass accuracy, and mass resolution of viral

proteins with matrix-assisted laser desorption ionization are

dependent on sample preparation and complexity, as well as mas s spectrometric conditions. Capsid proteins often contain hydrophobic

regions that maintain the protein-protein associations. These

hydrophobic regions contribute to the nucleocapsid or coat structure ,

and cause aggregation in aqueous solution. Despite a high percentage

of organic content in the matrix solvent, no signal was observed from

virus sample and matrix alone.

EXAMPLE 5

Detection of MS2 Bacteriophage

The growth conditions for MS2 yield numerous amounts of

biomolecules released from lysed host bacterial cells. In addition, th e

optimal broth contains large quantities of salts, peptides, and proteins ,

which potentially suppress the signal of interest in the matrix -

assisted laser desorption ionization spectrum. Considering the MS 2

phage culture had an active concentration of 2.1 x l 0¹⁰ pfu/ml, which

translates to a minimum 1 femtomole of coat protein applied to th e

slide well, any signal suppression could lead to no observed peak of

interest. No signal was observed with prior application of

nitrocellulose, PVDF membranes, or C8 membranes with and without washing. Such surfaces sometimes have higher laser inten sity

thresholds for desorption of ions (12).

The dissociation of viral proteins have historically been

accomplished with urea (13, 14), acetic acid (15) or detergents ( 16 ,

17). Careful selection of matrix additives is necessary due to signal

suppression caused by the acidity /basicity, crystallization, and

desorption properties of the additives (12, 18).

Figure 2 demonstrates differences observed in matrix-

assisted laser desorption ionization spectra of semi-purified

preparations of a MS2 culture, which independently had acetic a n d

citric acid added to the solution. Citric acid yielded better mas s

accuracy, and mass resolution, but more doubly charged ions with

approximately the same laser intensity. Citric acid has three acidic

protons: one with a pKa lower than the single acidic proton of acetic

acid, and another equal to the acidic proton of acetic acid. S odium

citrate has been shown to enhance MALDI protein spectra from

buffered protein solutions laden with salts (19, 20).

Spectra of crude MS2 cultures did not require higher laser

intensity to observe the coat protein molecular ion; however, th e

resolution was poorer than spectra obtained from semi-purified

preparations (Figure 3). Conservation of the coat protein sequence means that mass difference from the calculated molecular weight

value of 13729 Da is due to natriation or kalionation. Spectra were

obtained with several matrices; however, spectral quality varied from

sample to sample of the crude phage solution. In addition,

phosphatidylethanolamine from the host E. coli cell walls were

observed with certain MALDI matrices.

EXAMPLE 6

Detection of Tobacco Mosaic Virus

The desorption of the tobacco mosaic virus from a tobacco

leaf presented the challenge of a host that could potentially inhibit

desorption of the protein of interest with the MALDI matrix, and that

contains an excess of molecules that could suppress MALDI detection.

Despite these complications, Figure 4 demonstrates the detection of

the tobacco mosaic virus directly from a leaf and a leaf extract

sample. The extract provided more intense (i.e. as much as lOx ion

current) detection of the TMV virus compared to direct detection

from a leaf piece. On the basis of the published complete nucleotide

sequence of the genomic RNA of the U2 strain of TMV, the expected

mass for the coat protein is 17,459 Da (21). The mass difference ma y

be attributed to mutations or salt adducts. Table 1 demonstrates th e

mass accuracy from a MALDI-TOF instrument, to which the

manufacturer attests a 0.1% mass accuracy from standards. The

addition of citric acid to infected leaf pieces provided more facile

detection of TMV than acetic acid. Dissociation of TMV coat proteins

has been associated with the disruption of carboxylate groups a t

subunit interfaces (22).

TABLE 1

Molecular Masses for Viral Proteins Measured without Isolation

Number of expected capsid Molecular masses Mass Phage or virus protein (molecules/virion) expected observed³ accuracy fm /z ) (m l?. ) i___

MS2 bacteriophage 180 13729 13784 ± 6^b 0.40 Tobacco mosaic virus 2130 17461 17464 ± 29 0.02

Venezuelan equine encephalitis 240 30941 31225 ±105 0.92

"average molecular mass; n EXAMPLE 7

Detection of Venezuelan Equine Encephalitis Virus.

From the semi-purified preparation of Venezuelan Equine

Encephalitis, Figure 5 displays the mass spectrum of the nucleocapsid

protein and an indication of the less glycosylated glycoprotein of th e

two transmembrane glycoproteins (El and E2). This virus sample i s

representative of a biological virus preparation, which contains

chemicals known to degrade matrix-assisted laser desorption

ionization spectra. Maximum laser intensity was needed to observe

the VEE capsid protein. While the capsid protein peak is clearly

visible at m/z 31220, the peak associated with the El glycoprotein a t

m/z 49950 is observed at a 2: 1 signal to noise ratio. Peak

suppression in UV-MALDI analysis of glycoproteins is a frequent

encountered problem due to properties of the sugar moiety.

Discussion

The present invention demonstrates that UV-MALDI can

be used to rapidly detect characteristic proteins of viruses an d

bacteriophages. Without pre-concentration and little or n o

purification, bacteriophage MS2, tobacco mosaic virus, an d Venezuelan equine encephalitis virus have been detected from a

variety of biological media within 3 minutes. Certain matrices an d

additives increase the probability of observing viral proteins ;

however, this does not limit the usefulness of this technique.

While MALDI mass spectrometry has been gaining

popularity as an analytical technique for a range of pure compounds ,

application of the MALDI mass spectrometry to samples in biological

media will expand the utilization of this technique. The concepts

applied in the present invention may be utilized for the rapid

screening of more complex viruses that contain a diverse number of

proteins .

The following references are cited herein.

( 1 ) Ember, L. R. C & E News 1996.

(2) Karas, M., et al, Anal. Chem. 1988, 60: 2299-2301.

(3 ) Jespersen, S., et al., J. Rapid Commun. Mass Spectrom. 1994, 8 :

58 1 -584.

(4) Beavis, R., et al, Proc. Natl. Acad. Sci., USA 1990, 87: 6873-6877.

(5 ) C. Petersen, J., et al., J. Virology 1974, 14: 740-744.

(6) Kinney, R., et al., Virology 1989, 170: 19-30.

(7 ) Grieder, F., et al., Virology 1995, 206: 994-1006. ( 8 ) Despeyroux, D., et al., J. Rapid Commun. Mass Spectrom. 1 996 ,

10: 937-941.

(9) Davis, J., et al, J. Mol. Biol. 1963, 6: 203-207.

( 10) Walkey, D. Applied Plant Virology; John Wiley & Sons, Inc.: New

York, 1985.

( 1 1 Kussman, M., et al, J. Mass Spectrom. 1997, 32: 593-601.

( 12 Worrall, T. A., et al., Anal. Chem. 1998, 70: 750-756.

( 13 Buzzell, A. J. Am. Chem. Soc. 1960, 82: 1636-1641.

( 14 Blowers, L. E., et al., J. Gen. Virol. 1982, 61 : 137-141.

( 15 Fraenkel-Conrat, H. Virology 1957, 4: 1-4.

( 16 Ohno, T., et al.,Virology 1977, 76: 429-432.

( 17 Wilson, T. M., et al., FEBS Letters 1976, 64: 285-289.

( 1 8 Bornsen, K., et al., Rapid Commun. Mass Spectrom. 1997, 1 1 :

603 609.

( 19 Bergman, A., et al., FEBS letters 1996, 397: 45-49.

(20 Walker, K. L., et al., Anal. Chem. 1995, 67: 4197-4204.

(21 Solis, I., et al., Virology 1990, 177: 553-558.

(22 Culver, J. N., et al., Virology 1995, 206: 724-730.

Any patents or publications mentioned in this

specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are

herein incorporated by reference to the same extent as if each

individual publication was specifically and individually indicated to

be incorporated by reference.

One skilled in the art will readily appreciate that th e

present invention is well adapted to carry out the objects and obtain

the ends and advantages mentioned, as well as those inherent therein .

The present examples along with the methods, procedures ,

treatments, molecules, and specific compounds described herein are

presently representative of preferred embodiments, are exemplary ,

and are not intended as limitations on the scope of the invention.

Changes therein and other uses will occur to those skilled in the art

which are encompassed within the spirit of the invention as defined

by the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1 . A method of detecting viral proteins from a complex

environment, comprising the steps of:

preparing a crude sample of said environment;

mixing said sample with an organic acid;

detecting viral proteins by utilizing mass spectrometry to

said sample.

2. The method of claim 1 , wherein said viral proteins

are structural proteins.

3. The method of claim 2, wherein said viral protein is

a capsid protein.

4. The method of claim 1 , wherein said crude sample is

selected from the group consisting of semi-purified and non-purified

preparations .

5. The method of claim 1 , wherein said organic acid is

selected from the group consisting of acetic acid and citric acid.

6. The method of claim 1 , wherein said organic acid is

premixed with the mass spectrometry matrix.

7. The method of claim 1 , wherein said detection is

accomplished in less than 3 minutes.

8. The method of claim 1 , wherein a femtomolar

concentration of said viral protein is detected.

9. The method of claim 1 , wherein said protein

detected is a bacteriophage protein.

10. The method of claim 1 , wherein said mas s

spectrometry is selected from the group consisting of fast atom

bombardment mass spectrometry, plasma desorption mas s

spectrometry, laser desorption mass spectrometry, matrix-assisted

laser desorption ionization time-of-flight mass spectrometry an d

electrospray mass spectrometry.